Glucocorticoids are lipid-soluble hormones that signal via the glucocorticoid receptor (GR), a ligand dependent transcription factor. Circulating glucocorticoids derive fromthe adrenals, but it is now apparent that paracrine glucocorticoid signaling occurs in multiple tissues. Effective local glucocorticoid concentrations and whether glucocorticoid delivery can be targeted to specific cell subsets are unknown. We used fluorescence detection of chromatin-associated GRs as biosensors of ligand binding and observed signals corresponding to steroid concentrations over physiological ranges in vitro and in vivo. In the thymus, where thymic epithelial cell (TEC)-synthesized glucocorticoids antagonize negative selection, we found that CD4+CD8+TCRhi cells, a small subset responding to self-antigens and undergoing selection, are specific targets of TEC-derived glucocorticoids and are exposed to 3-fold higher levels than other cells. These results demonstrated and quantitated targeted delivery of paracrine glucocorticoids. This approach may be used to assess in situ nuclear receptor signaling in a variety of physiological and pathological contexts.